Artificial recombinant substrate (rAGG 1) and native aggrecan to determine the proteolytic activity of ‘aggrecanase’ in cell culture systems

ABSTRACT

The invention relates to novel recombinant substrates for aggrecanase. In one embodiment, this substrate comprises the signal sequence of CD5, the FLAG-epitope for M1 monoclonal antibody detection, the interglobular domain of human aggrecan, the hinge region of human IgG1, the CH2 region of human IgG1 and the CH3 region of human IgG1. DNA sequences encoding the recombinant substrate are also provided, as are vectors and host cells containing said DNA. Various methods are provided for: monitoring aggrecanase activity, detecting new enzymatic cleavage sites, purifying aggrecanase chromatographically, and cloning the aggrecanase cDNA, screening for aggrecanase inhibitors; and for monitoring the onset or progression of osteoarthritis. A diagnostic aid containing the recombinant substrate is also provided.

This application is a divisional of application Ser. No. 08/784,512,filed Jan. 17, 1997, now U.S. Pat. No. 5,872,209.

BACKGROUND OF THE INVENTION

Mechanisms of proteoglycan breakdown in connective tissue are complexand involve multiple agents and pathways. Aggrecan is the largeaggregating chondroitin sulfate proteoglycan of cartilage. See, forexample, Doege, et al. J. Biol. Chem. 266:894 (1991); GenBank/EMBLAccession Number M55172 (human aggrecan). In studies investigating thecatabolism of aggrecan, experimental systems used have includedmonolayer cultures of primary chondrocytes from established chondrocytecell lines (Hughes C E, et al., Biochem. J. (1995) 305:799-804; Lark M Wet al., J. Biol. Chem. (1995) 270(6):2550-2556) and explant culturesusing cartilage from a variety of anatomical sites and animal species(Flannery C R, et al. J. Biol. Chem. (1992) 267:1008-1014; Sandy J D, etal. Biochim Biophys Acta (1978) 543:536-44; Tyler J A, Biochem. J. 1985;225:493-507). The addition of cytokines such as IL-1 and TNF have beenextensively used as agents which promote the degradation of theextracellular matrix (Hughes C E et al., supra (1995) Morales T I, etal. Arch Biochem Biophys (1992) 293(1):79-84; Fosang A J, et al. Matrix(1991) 11:17-24).

In particular, these two cytokines have been shown to target thecatabolism of aggrecan.

Several studies have now lead to a number of important discoveries whichhave defined specific cleavage sites along the protein core of aggrecan(Ilic M Z, et al. Arch Biochem Biophys (1992) 294(1):115-22; Loulakis P,et al. Putative site(s) of enzymic cleavage. Biochem J (1992)284:589-593; Sandy J D, et al. J. Biol. Chem. (1991) 266:8683-8685). Intotal there appear to be at least seven cleavage sites, and amino acidsequence analysis of cartilage proteoglycan breakdown products havedefined two major sites of proteolytic cleavage in aggrecan which occurwithin the interglobular domain (IGD) between amino acid residuesAsn³⁴¹-Phe³⁴² (“AF”) and Glu³⁷³-Ala³⁷⁴ (“EA”) (human sequenceenumeration). Doege K J, et al. J. Biol. Chem. (1991) 266:894-902. TheAF cleavage site generates a C-terminal catabolic fragment (ending withthe C-terminal sequence DIPEN) consisting of the 50-60 kDa G1 domainthat remains in the tissue complexed to hyaluronate (Flannery C R, etal., 1992). In recent studies Fosang et al. (Trans. Orthop. Res. Soc.(1995)20:4) have also identified N-terminal fragments (beginning withthe N-terminal sequence FFG) from this cleavage site in synovial fluidsfrom patients diagnosed with a variety of different arthritides (jointdisease). Similarly, Witt et al (Trans. Orthop. Res. Soc. 1995; 20:122)have found these glycosaminoglycan-containing N-terminal fragments inmedia samples from control and IL-1 stimulated porcine explant cultures.

The EA cleavage site produces glycosaminoglycan-containing N-terminalfragments (ARG . . . ) that appear as the major aggrecan degradationproducts isolated from synovial fluid of patients with arthritis(Lohmander LS, et al. Arth. Rheum. (1993) 36:1214-1222). TheseN-terminal fragments are also found in media from cartilage explantcultures treated with IL-1 or retinoic acid (Hughes C E, et al., 1995;Sandy J D, et al., 1991). A recent study (Lark M W, et al., 1995)identified the C-terminal fragment (ending with the C-terminal sequenceEGE) in rat chondrosarcoma cells treated with retinoic acid.

The proteolytic activity responsible for this Glu³⁷³-Ala³⁷⁴ cleavage hasnot been identified but it appears to have specificity for Glu-Xaapeptide bonds where Xaa is Ala, Gly or Leu. This activity has beentermed “aggrecanase.” Fosang A J, et al. J. Biol. Chem. (1992)267:19470-19474). As used in this specification, “aggrecanase” means apolypeptide (or polypeptides) that will catalyze cleavage of suchGlu-Xaa peptide bonds in aggrecan. Although the sites of cleavage withinthe molecule have been well characterized, many of the agentsresponsible for generating the large number of different proteoglycandegradation products are still unidentified. It is believed that morethan one enzyme is responsible for degradation of proteoglycans. Culturesystems have been manipulated with a variety of agents that enhanceproteoglycan catabolism, in an effort to discover the agent(s)responsible for its breakdown. However, these studies have beenunsuccessful in defining any specific agent responsible for thedegradation of aggrecan. Flannery C R, et al. Trans. Orthop. Res. Soc.(1993). Nonetheless, experimental data using purified aggrecan andmodified aggrecan as a substrate for purified enzyme preparations hasyielded some information on the specific cleavage sites of the enzymesinvolved in proteoglycan degradation. However, this in vitro work hasnot definitively ascertained the mechanism or identity of agentsinvolved in the degradation of aggrecan in cartilage. Fosang A J, et al.(1992); Fosang A J, et al. J. Biol. Chem. (1991) 266:15579-15582; FosangA J, et al., Biochem. J. (1994) 304:347-351.

In previous studies (Hughes C E, et al., supra, (1995); Hughes C E, etal. J. Biol. Chem. (1992) 267:16011-16014) a number of monoclonalantibodies have been developed that are specific for the products(proteoglycan aggregate catabolites) that have been cleaved by specificproteinases. These antibodies have proved useful as tools in studyingthe mechanisms of breakdown of proteoglycan in cartilage explant culturesystems, by allowing monitoring of specific cleavage products.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides an isolated recombinantpolypeptide substrate for aggrecanase. In another embodiment, theinvention provides a recombinant substrate for aggrecanase thatcomprises the following components, beginning with the N-terminus andending with the C-terminus:

a) the signal sequence of CD5;

b) the FLAG-epitope;

c) the interglobular domain of human aggrecan;

d) the hinge region of human IgG1;

e) the CH2 region of human IgG1; and

f) the CH3 region of human IgG1.

In another embodiment, the invention relates to a recombinant substratefor aggrecanase that comprises the amino acid sequence as set forth inFIG. 6 (SEQ. ID NO. 3). In yet another embodiment, the invention relatesto a recombinant substrate that comprises a portion of the amino acidsequence as set forth in FIG. 6 (SEQ. ID NO. 3).

In a further embodiment, the invention relates to a recombinantsubstrate for aggrecanase that comprises the amino acid sequence as setforth in FIG. 6 (SEQ. ID NO. 3) and wherein amino acid 34 is mutated toAla. In yet a further embodiment, the invention relates to a recombinantsubstrate for aggrecanase that comprises a portion of the amino acidsequence as set forth in FIG. 6 (SEQ. ID NO. 3) and wherein amino acid34 is mutated to Ala.

In another embodiment, the invention relates to an isolated DNA sequenceencoding a recombinant substrate for aggrecanase. In a furtherembodiment, one such DNA sequence comprises the nucleotide sequence ofnucleotides 2350 to 4114 of FIG. 7 (SEQ. ID NO. 4). In anotherembodiment, another such DNA sequence comprises a portion of thenucleotide sequence of nucleotides 2350 to 4114 of FIG. 7 (SEQ. ID NO.4). In yet a further embodiment, the invention relates to a DNA sequenceencoding a substrate for aggrecanase, wherein said DNA sequencehybridizes under stringent conditions with the nucleotide sequence ofnucleotides 2350 to 4114 of FIG. 7 (SEQ. ID NO. 4).

The invention also relates to a vector comprising a DNA sequenceencoding a recombinant substrate for aggrecanase. In another embodiment,the invention relates to a host cell comprising such a vector. In afurther embodiment, the invention relates to a vector comprising thenucleotide sequence set forth in FIG. 7 (SEQ ID NO:4).

In still another embodiment, the invention relates to a cell culturesystem (or method) for monitoring aggrecanase activity in a samplecomprising:

(a) mixing freshly isolated chondrocyte cells and a recombinantsubstrate for aggrecanase;

(b) incubating the reaction mixture of step (a); and

(c) detecting the presence or absence of aggrecanase activity in thereaction mixture,

wherein aggrecanase activity is determined by the presence ofaggrecanase peptide cleavage products.

In one embodiment, such a system is free of endogenous proteoglycans orother extracellular components.

In another embodiment, the presence or absence of cleavage products ismeasured in such a system by determining the presence of peptidecleavage products that react with monoclonal antibodies specific foraggrecanase cleavage products. In a further embodiment, the monoclonalantibody detects a peptide having an amino acid sequence ARGSV. In yetanother embodiment, the aggrecanase activity of the chondrocytes in sucha system is stimulated by adding retinoic acid to the reaction mixtureof step (a).

The invention further relates to a method for cloning aggrecanase cDNAcomprising:

(a) preparing a cDNA expression library from cells expressingaggrecanase;

(b) transfecting suitable cells with the library of step (a), whereinsaid cells express said cDNA;

(c) incubating the cells of step (b) with a recombinant substrate foraggrecanase; and

(d) detecting the presence of an aggrecanase cleavage product producedby a cell of step (c).

In still another embodiment, the invention relates to a method forscreening for an aggrecanase inhibitor, comprising:

(a) mixing freshly isolated chondrocyte cells and a recombinantsubstrate for aggrecanase;

(b) incubating the reaction mixture of step (a) in the presence orabsence of a putative aggrecanase inhibitor; and

(c) detecting the presence or absence of aggrecanase activity in thereaction mixture,

wherein aggrecanase activity is determined by the presence ofaggrecanase peptide cleavage products.

In still another embodiment, the invention relates to a method formonitoring the onset of osteoarthritis, said method comprising assayinga sample of biological fluid from a patient for the presence ofaggrecanase, wherein said aggrecanase activity is measured using asystem described above. In another embodiment, the invention relates tosuch a monitoring method wherein said biological fluid is selected fromthe group consisting of synovial fluid, urine, serum, and lymph fluid.

In another embodiment, the invention relates to a method for monitoringthe progression of osteoarthritis, said method comprising assaying asample of biological fluid from a patient suffering from osteoarthritisfor the presence of aggrecanase, wherein said aggrecanase activity ismeasured using a system as described above. In another embodiment, theinvention relates to such a monitoring method wherein said biologicalfluid is selected from the group consisting of synovial fluid, urine,serum, and lymph fluid. In still another embodiment, the inventionrelates to such a monitoring method, wherein said method is used tofollow disease progression to determine the effectiveness of therapeutictreatment.

Another embodiment of the present invention is a diagnostic aidcomprising a recombinant substrate for aggrecanase and antibodies forthe detection of aggrecanase cleavage products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the overall structure of rAGG-1. rAGG-1 consists of: Signalsequence of CD5 (SS); FLAG-epitope for M1 monoclonal antibody detection(FLAG); interglobular domain of human aggrecan (IGD); hinge region ofhuman IgG1 (H); CH2 region of human IgG1 (CH2); CH3 region of human IgG1(CH3).

FIG. 2 shows detection of rAGG-1 expression in COS cells with monoclonalantibody M1.

FIGS. 3A-3C show SDS-PAGE of purified rAGG-1. Lane A: Coomassiestaining; lane B: Western blot detection with monoclonal antibody M1 andanti-human IgG antiserum, lane C.

FIGS. 4A-4C show BC-3 reactivity of rAGG-1 in cell culture medium fromrat chondrosarcoma cells. Lane A: rAGG-1 from a culture of unstimulatedrat chondrosarcoma cells is not detected with BC-3 monoclonal antibodyon a western blot, whereas rAGG-1 from a culture of retinoic acidstimulated cells is detected as shown in lane B. Lane C: A similar blot,after reprobing with monoclonal antibody M1. The BC-3 reactive fragmentis approximately 5.6 kD smaller than the original 72 kD band, indicatingthe cleavage of rAGG-1 at the “aggrecanase” site.

FIG. 5 shows set up of a 96 well format screening assay (with pin lid)to detect “aggrecanase” activity.

FIGS. 6A and 6B show the amino acid sequence of the recombinantpolypeptide substrate rAGG-1—Amino acids 1-24: CD5 signal sequence;Amino acids 25-32:Flag-sequence; Amino acids 33-160: Human aggrecaninterglobular domain; Amino acids 161-164: Spacer sequence; Amino acids165-179: Hinge region of human IgG1; Amino acids 180-289: CH2 region ofhuman IgG1; Amino acids 290-396: CH3 region of human IgG1.

FIGS. 7A-7C show the nucleotide sequence of vector prAGG-1-IGG(pCDM8-rAGG-1) for eucaryotic expression of rAGG-1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a recombinant polypeptide substrate(rAGG1) and native aggrecan for the study of the proteolytic activity of“aggrecanase” in cell culture systems. As used in this specification, a“recombinant polypeptide substrate” for aggrecanase means a polypeptidethat can be cleaved by aggrecanase and that is a non-naturally occurringprotein. This invention provides, for the first time, the production anduse of an recombinant polypeptide substrate for the study of‘aggrecanase’ activity. This invention further refines culture systemsthat will help facilitate the identification of the agents responsiblefor the cleavage of aggrecan at the ³⁷⁴ARGSVI . . . site in the IGD ofaggrecan.

The present application describes the development of an in vitro cellculture system (or method) that has enabled study of the activity of“aggrecanase” against a recombinant polypeptide substrate in a cellculture system that is free of inherent endogenous proteoglycans andother extracellular components. As used in this specification, “free of”is used to mean that such proteoglycans and other components are presentin a concentration low enough so that they do not significantly competewith the recombinant substrate in the aggrecanase assay. An “endogenousproteoglycan,” as used in this specification, refers to thoseproteglycans produced by the cells in a cell culture system. “Otherextracellular components” is used to refer to large molecular weightmolecules made by cells in such a cell culture system that are secretedinto the medium or retained on the cell surface. Examples of suchcomponents are proteoglycans and high molecular weight proteins andglycoproteins. The use of previously characterized neoepitope antibodiesto key cleavage sites in the IGD of aggrecan facilitated the monitoringof products generated by the proteolytic action of “aggrecanase” in thissystem.

Therefore, an embodiment of the present invention is a recombinantpolypeptide substrate for aggrecanase in vitro testing systems. In oneembodiment, there is provided a recombinant polypeptide substrate whichcontains a group of structural elements comprising

a) the signal sequence of CD5,

b) the FLAG-epitope,

c) the interglobular domain of human aggrecan,

d) the hinge region of human IgG1,

e) the CH2 region of human IgG1 and

f) the CH3 region of human IgG1.

The FLAG epitope can be identified by the M1 monoclonal antibody.Regarding element (c), the interglobular domain of aggrecan from anon-human species can also be used. Full length cDNAs have been reportedfor the following species: rat (GenBank J03485; Doege, et al. J. Biol.Chem. 262:17757 (1987)) and mouse (GenBank U22901; Watanabe, et al.Biochem. J. 308:433 (1995)). Partial cDNA sequences have been reportedfor the following species: chicken (Sai et al. PNAS USA 83:5081 (1986);Kruger et al. J. Biol. Chem. 265:12088 (1990) and bovine (GenBankY00319, J05028).

In one embodiment of the invention, these elements form a fusionprotein, beginning with the N-terminus, comprising elements a, b, c, d,e, and f (a-b-c-d-e-f).

Another embodiment of the present invention is a recombinant substratewhich has the amino acid sequence of FIG. 6 (SEQ. ID NO. 3). In yetanother embodiment, the invention provides such a substrate having aportion of the amino acid sequence of FIG. 6 (SEQ. ID NO. 3). In yetanother embodiment, the invention provides a substrate having the aminoacid sequence of FIG. 1 (SEQ. ID NO. 3), wherein the amino acid 34 ismutated to Ala. In still another embodiment, the invention provides sucha mutated substrate having a portion of the amino acid sequence of FIG.1 (SEQ. ID NO. 3), wherein the amino acid 34 is mutated to Ala.

Another embodiment of the present invention is a nucleotide sequence,such as DNA, encoding a recombinant polypeptide substrate according tothe invention. In one embodiment, the invention provides a DNA sequencehaving the nucleotide sequence of nucleotides 2350 to 4114 of FIG. 7(SEQ. ID NO. 4). In another embodiment, the invention provides a DNAsequence having a portion of the DNA sequence of FIG. 7 (SEQ. ID NO. 4).In yet another embodiment, the invention provides the nucleotidesequence of nucleotides 2350 to 4114 of FIG. 7 (SEQ. ID NO. 4), whereinnucleotide 2448 is mutated to C, nucleotide 2450 is mutated to C andnucleotide 2451 is mutated to A. In yet another embodiment, theinvention provides a portion of the nucleotide sequence of nucleotides2350 to 4114 of FIG. 7 (SEQ. ID NO. 4), wherein nucleotide 2448 ismutated to C, nucleotide 2450 is mutated to C and nucleotide 2451 ismutated to A.

In still another embodiment, the invention provides a nucleotidesequence which hybridizes under stringent conditions with the DNAsequence shown in FIG. 7 (SEQ. ID NO. 4). Hybridization under stringentconditions according to the invention means hybridization at atemperature of 20 to 25° C., or about 20 to 25° C., under the meltingpoint (T_(m)) formed between a probe and its target, in a hybridizationsolution containing 6×SSC (or alternatively 6×SSPE), 0.5% SDS and 100μg/ml denatured, fragmented Salmon sperm DNA (Sambrook et al.,“Molecular Cloning-A Laboratory Manual”, Second Edition, 1989, ColdSpring Harbor Laboratory Press, Volume 2, chapter 9, pages 9.52-9.55,hereby incorporated by reference). The skilled artisan will recognizethat the T_(m) of a probe and its target may be estimated using theequation at the top of page 9.51 of Sambrook.

In other embodiments, the present invention provides a vector containinga DNA fragment comprising a nucleotide sequence as described above(encoding a recombinant polypeptide substrate for aggrecanase) and ahost cell containing said vector. Suitable vectors include, but are notlimited to pCDM8, pCDNAI, pCDNAIII, pEUK-C1, and pMAM. Suitable hostcells include, but are not limited to COS, COS-7, CHO, BHK, and HeLa.

In another embodiment, the invention provides a cell culture system formonitoring aggrecanase activity in a sample comprising (1) mixingfreshly isolated chondrocyte cells and a substrate according to theinvention; (2) incubating the reaction mixture; and (3) detecting thepresence or absence of aggrecanase activity in the reaction mixture,wherein aggrecanase activity is determined by the presence ofaggrecanase peptide cleavage products. The presence or absence ofcleavage products is measured by determining the presence of peptidecleavage products that react with monoclonal antibodies that arespecific for aggrecanase cleavage products. Aggrecanase cleavageproducts are fragments of the recombinant substrate, which have theaminoterminus ARGSV. A monoclonal antibody that is “specific for anaggrecanase cleavage product” will spefically detect such cleavageproducts. For example, antibody BC-3 detects the amino terminus, ARGSV,on aggrecan degradation products. Hughes, et al. Biochem. J. 305:799(1995). The skilled artisan will recognize that other monoclonalantibodies that are specific for aggrecanase cleavage products can bemade using procedures that are well known to the skilled artisan. See,for example, Hughes, et al. J. Biol Chem. 267:16011 (1992).

The skilled artisan will recognize that the length of the incubation ofthe reaction mixture containing substrate and cells will vary accordingto experimental conditions. Suitable reaction conditions include, butare not limited to the following procedure: Cells such as ratchrondrosarcoma cells or primary bovine chondrocytes are grown embeddedin FMS Seaplaque agarose in DMEM medium at about 37° C. at a density ofabout 2×10⁶ cells/ml in 24 well culture plates. 20μ recombinantsubstrate is added into the DMEM medium and the culture medium isbrought to about 10⁻⁶M retinoic acid. Culture are kept for 48-96 hoursat about 37° C. The medium will contain fragments of the recombinantsubstrate with the aminoterminus ARGSV, which can be quantified.

In one alternative embodiment, the cell culture system is free ofinherent endogenous proteoglycans and other extracellular components.Thus, this novel cell culture system allows monitoring of “aggrecanase”activity without the complication of endogenous aggrecan acting as asubstrate. In addition, freshly isolated chondrocytes can be used inthis method without the need to establish an endogenous extracellularmatrix to act as a substrate for any ‘aggrecanase’ activity. Prior artmethods relied on an established extracellular matrix for assaying“aggrecanase” activity, such as aggrecan G1-G2 domains from piglaryngeal cartilage. See Hughes, et al. J. Biochem. 305:799 (1995).

Newly developed culture systems according to the invention will proveuseful for further studies of the molecular mechanisms involved inaggrecan degradation by “aggrecanase.” It is also a novel experimentalapproach that for studies of the catabolism of other matrixmacromolecules (such as link protein, brevican, other proteoglycans andcollagens) given the availability of recombinant polypeptide substratesand neoepitope antibodies.

A further embodiment of the present invention is a method as describedabove, wherein the study of the activity of aggrecanase is performed bydetection of cleavage products by antibodies that are specific forcertain cleavage products.

Another embodiment of the present invention is a method for thedetection of new enzymatic cleavage sites in a recombinant polypeptidesubstrate, comprising using a recombinant substrate according to theinvention in a suitable system for measuring enzymatic activity. Thesubstrate is incubated with an enzyme, such as aggrecanase, undervarying conditions and the cleavage products are analyzed.Peptide-specific monoclonal antibodies such as BC-3 or BC14 are used todetermine the identity of cleavage products. Alternatively, cleavageproducts are purified using techniques well-known to the skilled artisanand are subjected to N-terminal sequence analysis.

Another embodiment of the present invention is a method for thepurification of aggrecanase by means of affinity chromatography using arecombinant substrate according to the invention, linked to an affinitymatrix.

In yet another embodiment, the recombinant substrate is used togetherwith the antibody BC-3 to monitor aggrecanase activity duringpurification of aggrecanase activity. Thus, enrichment of aggrecanase ina sample is measured by the ability of such a sample to catalyzebreakdown of a recombinant substrate according to the invention bymonitoring with BC-3 antibody. Suitable methods for such purificationinclude ion exchange chromatography, gel filtration chromatography, andother techniques that are well known to the skilled artisan.

In yet another embodiment, the invention provides a functional cloningsystem for the isolation of aggrecanase cDNA. In such a system, an“expression library” is plated on medium containing a recombinantpolypeptide substrate according to the invention. A suitable expressionlibrary is prepared from retinoic acid-stimulated rat chondrosarcomacells in the plasmid pCDM8. Cells, such as COS-7 cells, are transfectedwith this library and plated in 96 well culture plates. The recombinantsubstrate is added into the medium of the culture plates the plates areincubated for at least about 48 hours to allow for cleavage of thesubstrate by aggrecanase. Cleavage of substrate at an “aggrecanase” sitewill be indicated by reactivity of expression products with the BC-3antibody, and will indicate the presence of a clone containing cDNAencoding “aggrecanase” activity.

In yet another embodiment, the invention provides a method formonitoring the onset or progression of osteoarthritis, said methodcomprising assaying a sample of biological fluid, from a human suspectedor known to have osteoarthritis, for the presence of aggrecanase bymeans of the recombinant polypeptide substrate as described above,wherein said biological fluid is selected from the group consisting ofsynovial fluid, urine, serum, and lymph fluid. It is known that thepresence of “aggrecanase” activity in one or more of such fluids iscorrelated with the onset and progression of osteoarthritis andrheumatoid arthritis, with “aggrecanase” activity increasing as thedisease progresses.

Thus, an additional embodiment of the present invention is a“monitoring” method as described above, wherein said method is used tofollow disease progression to determine the effectiveness of atreatment. As the effectiveness of treatment increases, “aggrecanase”activity will decrease.

Another embodiment of the present invention is a method for thescreening for an aggrecanase inhibitor, comprising the following steps.Suitable cells, such as rat chondrosarcoma cells, are cultivated inagarose in 96 well plates in medium (such as DMEM) and stimulated withretinoic acid to induce expression of aggrecanase. The recombinantsubstrate is attached to the surface of plastic pins of plastic lids for96 well plates (such as NUNC TSP plates). The substrate is incubated inthe medium (with or without a potential aggrecanase inhibitor) for asuitable time to allow cleavage by aggrecanase (e.g., for about 48hours). The attachment of the substrate is achieved via binding thecarboxyterminal immunoglobulin domain of the substrate anti-human IgGantibodies non-specifically adsorbed to the plastic of the pins.ARGSV-neoepitopes produced by the aggrecanase action on the recombinantsubstrate is detected via immunodetection with an ARGSV-specificmonoclonal antibody (such as BC-3). The amount of bound BC-3 antibody isquantified via detection with goat anti-mouse IgG peroxidase antibodyand a suitable color substrate.

The skilled artisan will recognize that aggrecanase inhibitors willdecrease the formation of ARGSV-neopeptide, compared with controlconditions (no inhibitor present).

Thus, in another embodiment of the present invention, theabove-described method for monitoring aggrecanase activity is used tomeasure the amount of aggrecanase activity in a tissue or fluid samplefrom a patient. For example, the synovial fluid from the knee of apatient is placed into a 96-well plate and the bound substrate, asdescribed above, is added to the fluid samples. Aggrecanase activity isdetermined by immunodetection with BC-3 antibody.

Another embodiment of the present invention is a diagnostic aidcomprising a recombinant polypeptide substrate as described above andantibodies for the detection of aggrecanase cleavage products. Asdescribed above, following cleavage of the recombinant substrate,antibody, such as BC-3, can be added to quantify the amount ofaggrecanase activity.

The following examples are added to illustrate the present invention anddo not serve to limit the scope of the present invention.

EXAMPLES

Materials: Alkaline phosphatase-conjugated second antibody and substrateused in Western blot analysis were obtained from Promega as theProtoblot Western blot AP system (catalog no. W3920). Nitrocellulose(0.2-μm pore size) was obtained from Schleicher and Schuell. Monoclonalantibody M1 and Anti-FLAG M1 Affinity gel were both obtained from Kodak.The M1 antibody recognizes the eight-amino acid FLAG epitope. Anti-HumanIg monoclonal was obtained from Capell, Durham. Monoclonal antibodiesBC-3, 2-B-6 and 3-B-3 were prepared as ascitic fluid. Monoclonalantibody BC-3 recognizes the amino acid sequence ARGSV in human aggrecanand was prepared using the procedure described in Hughes, et al., 1995,Biochem. J. 305:799 (1995), hereby incorporated by reference. Monoclonalantibody 2-B-6 recognizes aggrecan proteoglycan core protein catabolitescontaining 4-sulfated oligosaccharide stubs and can be obtainedcommercially from ICN, order #69-622-2. See Caterson, et al., Biochem.Soc. Trans. 18:820 (1990), hereby incorporated by reference. Monoclonalantibody 3-B-3 recognizes unsaturated terminal disaccharide 6 which issulfated on the aggrecan core protein and 30 is commercially availablefrom ICN, order #69-621-2. See Couchman, et al. Nature 307:650 (1984).The Rx cell line was provided by Dr. Jim Kimura, Bone Research Center,Henry Ford Hospital, Detroit, Mich.

Example 1

Preparation of the rAGG1 Genetic Construct

The genetic construct for rAGG1 codes for the signal sequence of thelymphocyte glycoprotein T1/Leu-1 (CD5), the eight amino acid long FLAG™epitope (Prickett K S, Amberg D C, Hopp T P, Bio Techniques 1989;7:580-589), the 127 amino acid long interglobular domain of the humancartilage large aggregating proteoglycan aggrecan (³⁵⁰T-⁴⁷⁶G) (Doege KJ, et al., 1991), a two amino acid long glycine spacer and the humanIgG1 hinge, CH2 and CH3 constant regions, thus giving rise to a fusionprotein with an expected molecular mass of 41,059 daltons.

Aggrecan cDNA sequences encoding the interglobular domain were amplifiedby the reverse transcriptase polymerase chain reaction (RT-PCR) (BeverlyS M, Current Protocols in Molecular Biology. Canada: John Wiley andSons, 1992) with synthetic oligonucleotides complementary to sequencesflanking this region and human knee cartilage total RNA. Total RNA wasextracted from human cartilage tissue, obtained from joint replacementsurgery, according to Adams et al (Adams M E, Huang D Q, Yao L Y,Sandell L J, Anal. Biochemistry 1992; 202:89-95).

Oligonucleotides were designed to contain additional sequenceinformation regarding the FLAG epitope and the g-spacer and to allow thecreation of restriction enzyme cleavage sites at the 5′ and 3′extremities of the amplified cDNA segments to facilitate subsequentinsertion into the CD5-IgG1 expression vector, modified to contain a 3′NheI site. See Aruffo A, Stamenkovic I, Melcick M, Underhill C B, SeedB, Cell 1990; 61:1303-1313, hereby incorporated by reference. The primerencoding the FLAG epitope and the amino terminal start of theinterglobular domain and including an NheI site was synthesized with thefollowing sequence: 5′-CGC GGG GCT AGC CGA CTA CAA GGA CGA CGA TGA CAAGAC AGG TGA AGA CTT TGT GGA C (SEQ ID NO: 1). A reverse primer encodingthe carboxyterminal end of the interglobular domain, the G-spacer, asplice donor site containing a BamHI site had the sequence: 5′-CGC GGGGGA TCC CCT CCC CCT GGC AAA TGC GGC TGC CC (SEQ ID NO: 2). The PCRproduct was produced using human cartilage total RNA as template and wasdigested with NheI and BamHI and ligated to NheI and BamHI-cut vectorCD5-IgG (Aruffo A, et al., 1990). The sequence of the resulting vector,prAGG-1-IGG (pCDM8-rAGG-1), is given SEQ ID NO:4.

Example 2

Production and Purification of rAGG-1 in COS Cells

The prAGG-1-IGG construct was transfected into COS cells viaDEAE-dextran (Hollenbaugh D, Aruffo A, Current protocols in molecularbiology. Canada: John Wiley and Sons, 1994). Twelve hours aftertransfection, cells grown in DMEM, 10% FCS, were trypsinized, seededonto fresh dishes and allowed to grow for five days. On the third day,fresh media and 10% FCS was added. Supernatants were harvested,centrifuged to remove nonadherent cells and debris, pooled and stored at4° C. Fusion proteins were affinity purified via anti-FLAG™ M1 affinitygel (Kodak, New Haven) according to the manufacturer's protocol. Usuallythe yield was 1-5 μg fusion protein per ml of culture supernatant.

Example 3

Characteristics of the Recombinant IGD Construct

Transfection of the recombinant IGD construct into COS cells resulted inthe cellular expression and subsequent secretion of the recombinant IGDproduct into the media. The use of an anti-FLAG M1 affinity gelfacilitated its purification from transfected cell media. One liter oftransfected COS cell media yielded 1-5 mg of recombinant IGD substrate.Under non-reducing conditions this recombinant IGD substrate formedlarge molecular weight aggregates due to the presence of an unevennumber of cysteine residues in the immunoglobulin domain of themolecule. This aggregate formation facilitated immobilization of therecombinant IGD substrate in the agarose cultures. Under reducingconditions the recombinant IGD construct occurred as two molecularweight components having estimated Mr of 72 kDa and 39 kDa, as seenafter separation on 10% SDS-PAGE gels. The reasons for the occurrence oftwo forms of the recombinant IGD substrate are currently unknown,however, both the anti M1 monoclonal (recognizing an epitope in the FLAGregion of the construct) and the anti Ig monoclonal (recognizing anepitope in the immunoglobulin region of the construct) immunolocatedboth forms of the recombinant IGD construct. Densitometric analysis of aCoomassie stained gel of the separated recombinant IGD construct showedthat the ratio of the 72 kDa to the 39 kDa band was approximately 10:1.

Example 4

Culture of Rat Chondrosarcoma Cells in Agarose in the Presence orAbsence of Retinoic Acid for Studies of the Catabolism of theRecombinant IGD Substrate and Native Aggrecan

Rat chondrosarcoma cells (cell line: Swarm rat chondrosarcoma cell lineRx) were plated in 75 cm² flasks and maintained in DMEM media containing5% FCS and 50 μg/ml gentamicin. Confluent monolayers in 75 cm² flaskswere harvested by trypsinization (0.25% trypsin in DMEM containing EDTA)for 15-20 minutes at 37° C. with agitation followed by digestion in0.05% collagenase in DMEM for 1-2 hours. The cells were then resuspendedin DMEM at 4×10⁶ cells/ml. 24 well culture plates were ‘coated’ (200μl/well) with a 1% (w/v) solution of FMC Seaplaque agarose in DMEM andthe agarose solidified by incubation at 4° C. for 30 minutes (AydelotteM B, Juettner K E, Conn. tissue Res. 1988; 18:205-222). The plates werethen equilibrated to 37° C.

The rat chondrosarcoma cell suspension (described above) was dilutedwith a 2% solution of Seaplaque agarose in DMEM such that the final cellconcentration was 2×10⁶ cells/ml. 200 μl (0.4×10⁶ cells) of the agarosecell suspension was laid over the previously prepared agarose plugs andimmediately following this, 50 μg of recombinant IGD substrate (rAGG1)or native bovine aggrecan (A1D1) was added to the appropriate wells andmixed by agitation. The plates were then incubated at 4° C. for 15minutes to solidify the agarose. Experimental media (with or withoutretinoic acid) was then added to triplicate wells containing therecombinant IGD construct, native bovine aggrecan (A1D1) and cells aloneas follows: Control cultures, DMEM+gentamicin (50 μg/ml) and treatedcultures, DMEM+gentamicin (50 μg/ml)+10⁻⁶ M retinoic acid (Hughes C E,et al., 1995). Agarose cell cultures were then maintained at 37° C. in5% CO₂ for 96 hours after which media and agarose-cell matrix extractswere further analyzed. Upon stimulation with retinoic acid, these ratchondrosacroma cells (RX) are known to produce “aggrecanase,” asevidenced by the ability of RA-stimulated cells to degrade aggrecan. SeeLark et al., J. Biol. Chem. 270:2550 (1995, hereby incorpoared byreference.

Example 5

Analysis of Experimental Media From Control and Retinoic Acid StimulatedAgarose Cultures

Media from the recombinant IGD substrate cultures and the culturescontaining cells alone were dialyzed exhaustively against deionized H₂O(“d.H₂O”), lyophilized and reconstituted in an equal volume of SDS-PAGEsample buffer containing 10% (v/v) mercaptoethanol. Media samples fromthe A1D1 cultures and the cultures containing cells alone were dialyzedinto 0.1M Tris, 50 mM sodium acetate, pH 6.5 and deglycosylated usingmethods previously described (Hughes et al., 1995, supra.). Native A1D1was deglycosylated to remove any potential BC-3 epitopes that wereexposed in A1D1 prior to treatment with aggrecanase. Deglycosylation ofa recombinant substrate according to the invention is not necessary.

The samples were then dialyzed exhaustively against d.H₂O, lyophilizedand reconstituted in an equal volume of SDS-PAGE sample buffer. Samples(equal volumes/well) were then subjected to SDS-PAGE, transferred tonitrocellulose and immunolocated with either polyclonal or monoclonalantibodies using procedures described below.

Example 6

Extraction of Agarose-cell Matrix From Control and Retinoic AcidStimulated Cultures

Agarose plugs from recombinant IGD substrate cultures, bovine A1D1cultures and cultures containing cells alone were extracted for 24 hoursat 4° C. with 4M guanidinium chloride containing the following enzymeinhibitors: 10 mM EDTA, 5 mM benzamidine hydrochloride, 0.1M6-aminohexanoic acid and 1 mM phenylmethanesulphonyl fluoride. Theresidue of the agarose gel was removed by centrifugation and thesupernatants were then processed for each of the substrates as describedabove. Samples (equal volumes/well) were then subjected to SDS-PAGE,transferred to nitrocellulose and immunlocated with polyclonal andmonoclonal antibodies using procedures described below.

Example 7

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analyses

Samples containing native recombinant IGD substrate and its cataboliteswere electrophoresed on 10% polyacrylamide slab gels in SDS usingprocedures described by Laemmli (Laemmli U K, Nature 1970; 227:680-685).After electrophoresis the fractionated proteins were electrophoreticallytransferred to nitrocellulose and immunolocated with anti-FLAGmonoclonal or anti human immunoglobulin or monoclonal antibody BC-3 (allat a dilution of 1:1000) using procedures previously described (Hughes CE, et al., 1995). Samples containing native bovine A1D1 and itscatabolites were electrophoresed on 3-15% SDS-PAGE gels and transferredto nitrocellulose membranes for immunolocation with a 1:1000 dilution ofmonoclonal antibodies 3-B-3; 2-B-6 and BC-3. In general, the immunoblotswere incubated with substrate for 5-15 min at room temperature toachieve optimum color development.

Example 8

Immunochemical Analysis of the IGD Recombinant Construct From AgaroseCultures Treated with and without Retinoic Acid

Recombinant IGD substrate was immobilized in agarose cell culturescontaining rat chondrosarcoma chondrocytes, with or without retinoicacid, for 96 hours. Analysis of the IGD recombinant substrate and itsmetabolites in the culture media was examined by SDS-PAGE and WesternBlot analysis. Immunolocation with anti M1 (recognizing an epitope inthe FLAG domain at the N-terminal of the recombinant polypeptidesubstrate) showed no differences in the pattern of rAGG-1 metabolitespresent in media from either control cultures or retinoic acid treatedcultures. However, immunolocation with anti Ig (recognizing an epitopein the immunoglobulin domain at the C-terminal of the recombinantpolypeptide substrate) showed the presence of an additional bandmigrating at 65 kDa suggesting partial catabolism of the 72 kDaundigested recombinant polypeptide substrate had occurred in thepresence of retinoic acid. This result indicates that the M1 epitope (atthe N-terminal) was cleaved from the 72 kDa form of the IGD recombinantpolypeptide substrate to generate a 65 kDa catabolite. The absence ofpositive immuunostaining for the 72 kDa band with anti M1 confirms thisconclusion.

In order to ascertain if this cleavage of the IGD construct wasoccurring at the ‘aggrecanase’ site of the recombinant polypeptidesubstrate media samples of chondrocyte cultures treated with and withoutretinoic acid were examined by immunoblotting with monoclonal antibodyBC-3 (anti ARGSV). Immunolocation with BC-3 showed no reactivity incultures without retinoic acid and only one immunopositive band at 65kDa in the retinoic acid treated cultures. This result indicates that“aggrecanase” catabolism of the 72 kDa form of the IGD recombinantpolypeptide substrate has occurred in the presence of retinoic acid.However, there was no evidence that the 39 kDa isoform had beencatabolized (as described in Example 3). Absence of immunostaining withBC-3 for positive 39 kDa form catabolites maybe due to these catabolitesbeing present in lower concentrations since there was ten times more ofthe 72 kDa form in the rAGG-1 preparation than the 39 kDa form. In orderto confirm that the 65 kDa catabolite was derived from the 72 kDa formof rAGG-1 the BC-3 immunoblot was reprobed with the anti M1 antibody. M1immunodetected the uncleaved, parental peptide binds of 72 and 39 kD,and thus shows unequivocally that the BC-3 reactive cleavage product hasa smaller molecular mass that the parental 72 kD band.

The results obtained with the recombinant IGD construct demonstrate thatthere is not a need for a G1 and/or a G2 domain on the IGD of aggrecanfor cleavage at the ‘aggrecanase’ site. Reactivity with theaggrecanase-cleavage site-specific antibody BC-3 shows that therecombinant substrate rAGG-1 is cleaved at the aggrecanase site. Sincethis fusion protein does not contain a G1 or G2 domain, those domainsare not essential for aggrecanase to be able to cleave rAGG-1 at theaggrecanase cleavage site. Furthermore, there appears to be norequirement for keratan sulphate chains within the IGD which may giverigidity to this region in the native aggrecan molecule, because therAGG-1 construct showed no positive staining on Western blot analysiswith an anti-keratan sulphate antibody 5-D-4.

Example 9

Analysis of Native Bovine A1D1 From Agarose Cultures Treated with andwithout Retinoic Acid

The use of a native substrate in this culture system was also evaluatedto compare it with recombinant polypeptide substrate. Native bovineaggrecan (A1D1) was mixed with agarose containing rat chondrocytestreated with and without retinoic acid. Agarose cell cultures, withoutadded bovine A1D1, were also analyzed in order to assay for anycontribution of endogenous proteoglycan (potential substrate)synthesized during the 96 hours of culture.

When media samples were immunolocated with monoclonal antibody 2-B-6 nostaining was seen in media taken from agarose cultures containing cellsonly. This result shows that there was no significant contribution ofnewly synthesized endogenous substrate in the culture period.

Cultures containing exogeneously added bovine aggrecan substratemaintained with and without retinoic acid show the predictable array ofprotein cores, ‘ladders’, as previously described by several studies(Hughes C E, et al., supra (1995); Ilic M Z, et al. Biochem. Internat.1990; 21:977-986).

In contrast, immunostaining, using antibody BC-3, for the‘aggrecanase’-generated aggrecan catabolite with the N-terminal sequenceARGSV . . . was the only fragment observed in the cultures that weresubjected to retinoic acid treatment. As stated above, retinoic acidstimulates “aggrecanase” activity in this culture system. The relativemolecular weight of this BC-3 positive aggrecan catabolite was ˜160 kDaand is similar to that observed in other culture systems (Hughes C E, etal., 1995).

Media and extract samples from cultures with both the recombinantpolypeptide substrate and the native bovine aggrecan were also probedwith a newly developed antibody (BC-14) that recognizes the N-terminalneoepitope sequence F³⁴²FGVG . . . that is formed after Matrixmetalloproteinase (MMP) cleavage of human aggrecan that is generated byproteolysis of aggrecan IGD with MMP's 1, 2, 3, 7, 8 or 9.Immunoblotting with BC-14 was negative in both culture systems. Thisresult indicates that catabolism of the IGD at this site was notoccurring, a finding consistent with that reported from otherlaboratories (Lark M W, et al., 1995).

Example 10

Synthesis and Proteolysis of Mutated rAGG-1 Substrate

For further studies, the recombinant polypeptide substrate rAGG-1mut wasemployed, a version of our fusion protein rAGG-1, in which an alternatesplice donor site at the aminoterminal end of the IGD was mutated toprevent alternative splicing. The parent construct rAGG-1 of rAGG-1mutcontains a FLAG epitope as aminoterminal tag, the IGD of the humanaggrecan, and the constant region of a human IgG molecule as acarboxyterminal tag. The amino acid sequence of rAGG-1mut differs onlyin position 34 from the amino acid sequence of rAGG-1: rAGG-1mutcontains a Ala 34, while rAGG-1 contains a Gly 34. The nucleotidesequence of the DNA encoding rAGG-1mut differs in three positions fromthe DNA encoding rAGG-1: 2448: A to C, 2450: G to C and 2451: T to A.

Upon transient transfection, rAGG-1mut was secreted by COS cells as afusion protein into the culture supernatant and ran as a 72 kD bandunder reducing conditions and as a 140 kD band under non reducingconditions, probably reflecting a dimerisation due to the presence of anunpaired cysteine in the hinge region of the human immunoglobulincomponent at the C-terminal of the construct.

Upon stimulation with retinoic acid (RA), agarose embedded cells of therat chondrosarcoma cell line RX produce aggrecanase. rAGG-1mut iscatabolised at the aggrecanase site as has been described for rAGG-1,which is shown through the immunodetection with the monoclonal antibodyBC-3. The size of 66 kD of this catabolic product is in agreement withthe predicted loss of 5.8 kD after cleavage at the aggrecanase site,A³⁷⁴RGSV.

Priority application, European Application No. 96100682.2, filed Jan.18, 1996, including the specification, drawings, claims and abstract, ishereby incorporated by reference.

4 58 base pairs nucleic acid single linear DNA (genomic) unknown exon1..58 1 CGCGGGGCTA GCCGACTACA AGGACGACGA TGACAAGACA GGTGAAGACT TTGTGGAC58 38 base pairs nucleic acid single linear DNA (genomic) unknown exon1..38 2 CGCGGGGGAT CCCCTCCCCC TGGCAAATGC GGCTGCCC 38 396 amino acidsamino acid single linear protein unknown Protein 1..396 3 Met Pro MetGly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly 1 5 10 15 Met LeuVal Ala Ser Val Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys 20 25 30 Thr GlyGlu Asp Phe Val Asp Ile Pro Glu Asn Phe Phe Gly Val Gly 35 40 45 Gly GluGlu Asp Ile Thr Val Gln Thr Val Thr Trp Pro Asp Met Glu 50 55 60 Leu ProLeu Pro Arg Asn Ile Thr Glu Gly Glu Ala Arg Gly Ser Val 65 70 75 80 IleLeu Thr Val Lys Pro Ile Phe Glu Val Ser Pro Ser Pro Leu Glu 85 90 95 ProGlu Glu Pro Phe Thr Phe Ala Pro Glu Ile Gly Ala Thr Ala Phe 100 105 110Ala Glu Val Glu Asn Glu Thr Gly Glu Ala Thr Arg Pro Trp Gly Phe 115 120125 Pro Thr Pro Gly Leu Gly Pro Ala Thr Ala Phe Thr Ser Glu Asp Leu 130135 140 Val Val Gln Val Thr Ala Val Pro Gly Gln Pro His Leu Pro Gly Gly145 150 155 160 Gly Asp Pro Glu Glu Pro Lys Ser Cys Asp Lys Thr His ThrCys Pro 165 170 175 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser ValPhe Leu Phe 180 185 190 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgThr Pro Glu Val 195 200 205 Thr Cys Val Val Val Asp Val Ser His Glu AspPro Glu Val Lys Phe 210 215 220 Asn Trp Tyr Val Asp Gly Val Glu Val HisAsn Ala Lys Thr Lys Pro 225 230 235 240 Arg Glu Glu Gln Tyr Asn Ser ThrTyr Arg Val Val Ser Val Leu Thr 245 250 255 Val Leu His Gln Asp Trp LeuAsn Gly Lys Glu Tyr Lys Cys Lys Val 260 265 270 Ser Asn Lys Ala Leu ProAla Pro Ile Glu Lys Thr Ile Ser Lys Ala 275 280 285 Lys Gly Gln Pro ArgGlu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 290 295 300 Asp Glu Leu ThrLys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 305 310 315 320 Phe TyrPro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 325 330 335 GluAsn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 340 345 350Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 355 360365 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 370375 380 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 5337base pairs nucleic acid single linear DNA (genomic) unknown exon 1..53374 GGCGTAATCT GCTGCTTGCA AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG 60GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA 120AATACTGTCC TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG 180CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG 240TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA 300ACGGGGGGTT CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC 360CTACAGCGTG AGCATTGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT 420CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC 480TGGTATCTTT ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA 540TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCAAGCTA GCTTCTAGCT 600AGAAATTGTA AACGTTAATA TTTTGTTAAA ATTCGCGTTA AATTTTTGTT AAATCAGCTC 660ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT AAATCAAAAG AATAGCCCGA 720GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA CTATTAAAGA ACGTGGACTC 780CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC CGCCCACTAC GTGAACCATC 840ACCCAAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG 900GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA 960GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC 1020CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TACTATGGTT GCTTTGACGA 1080GCACGTATAA CGTGCTTTCC TCGTTGGAAT CAGAGCGGGA GCTAAACAGG AGGCCGATTA 1140AAGGGATTTT AGACAGGAAC GGTACGCCAG CTGGACCGCG GTCTTTCTCA ACGTAACACT 1200TTACAGCGGC GCGTCATTTG ATATGATGCG CCCCGCTTCC CGATAAGGGA GCAGGCCAGT 1260AAAAGCATTA CCCGTGGTGG GGTTCCCGAG CGGCCAAAGG GAGCAGACTC TAAATCTGCC 1320GTCATCGACT TCGAAGGTTC GAATCCTTCC CCCACCACCA TCACTTTCAA AAGTCCGAAA 1380GCTGCTCCCT GCTTGTGTGT TGGAGGTCGC TGAGTAGTGC GCGAGTAAAA TTTAAGCTAC 1440AACAAGGCAA GGCTTGACCG ACAATTGCAT GAAGAATCTG CTTAGGGTTA GGCGTTTTGC 1500GCTGCTTCGC GATGTACGGG CCAGATATAC GCGTTGACAT TGATTATTGA CTAGTTATTA 1560ATAGTAATCA ATTACGGGGT CATTAGTTCA TAGCCCATAT ATGGAGTTCC GCGTTACATA 1620ACTTACGGTA AATGGCCCGC CTGGCTGACC GCCCAACGAC CCCCGCCCAT TGACGTCAAT 1680AATGACGTAT GTTCCCATAG TAACGCCAAT AGGGACTTTC CATTGACGTC AATGGGTGGA 1740CTATTTACGG TAAACTGCCC ACTTGGCAGT ACATCAAGTG TATCATATGC CAAGTACGCC 1800CCCTATTGAC GTCAATGACG GTAAATGGCC CGCCTGGCAT TATGCCCAGT ACATGACCTT 1860ATGGGACTTT CCTACTTGGC AGTACATCTA CGTATTAGTC ATCGCTATTA CCATGGTGAT 1920GCGGTTTTGG CAGTACATCA ATGGGCGTGG ATAGCGGTTT GACTCACGGG GATTTCCAAG 1980TCTCCACCCC ATTGACGTCA ATGGGAGTTT GTTTTGGCAC CAAAATCAAC GGGACTTTCC 2040AAAATGTCGT AACAACTCCG CCCCATTGAC GCAAATGGGC GGAATTCCTG GGCGGGACTG 2100GGGAGTGGCG AGCCCTCAGA TGCTGCATAT AAGCAGCTGC TTTTTGCCTG TACTGGGTCT 2160CTCTGGTTAG ACCAGATCTG AGCCTGGGAG CTCTCTGGCT AACTAGAGAA CCCACTGCTT 2220AAGCCTCAAT AAAGCTTCTA GAGATCCCTC GACCTCGAGA TCCATTGTGC TCTAAAGGAG 2280ATACCCGGCC AGACACCCTC ACCTGCGGTG CCCAGCTGCC CAGGCTGAGG CAAGAGAAGG 2340CCAGAAACCA TGCCCATGGG GTCTCTGCAA CCGCTGGCCA CCTTGTACCT GCTGGGGATG 2400CTGGTCGCTT CCGTGCTAGC CGACTACAAG GACGACGATG ACAAGACAGG TGAAGACTTT 2460GTGGACATCC CAGAAAACTT CTTTGGAGTG GGGGGTGAGG AGGACATCAC CGTCCAGACA 2520GTGACCTGGC CTGACATGGA GCTGCCACTG CCTCGAAACA TCACTGAGGG TGAAGCCCGA 2580GGCAGCGTGA TCCTTACCGT AAAGCCCATC TTCGAGGTCT CCCCCAGTCC CCTGGAACCC 2640GAGGAGCCCT TCACGTTTGC CCCTGAAATA GGGGCCACTG CCTTCGCTGA GGTTGAGAAT 2700GAGACTGGAG AGGCCACCAG GCCCTGGGGC TTTCCCACAC CTGGCCTGGG CCCTGCCACG 2760GCATTCACCA GTGAGGACCT CGTCGTGCAG GTGACCGCTG TCCCTGGGCA GCCGCATTTG 2820CCAGGGGGAG GGGATCCCGA GGGTGAGTAC TAAGCTTCAG CGCTCCTGCC TGGACGCATC 2880CCGGCTATGC AGCCCCAGTC CAGGGCAGCA AGGCAGGCCC CGTCTGCCTC TTCACCCGGA 2940GGCCTCTGCC CGCCCCACTC ATGCTCAGGG AGAGGGTCTT CTGGCTTTTT CCCCAGGCTC 3000TGGGCAGGCA CAGGCTAGGT GCCCCTAACC CAGGCCCTGC ACACAAAGGG GCAGGTGCTG 3060GGCTCAGACC TGCCAAGAGC CATATCCGGG AGGACCCTGC CCCTGACCTA AGCCCACCCC 3120AAAGGCCAAA CTCTCCACTC CCTCAGCTCG GACACCTTCT CTCCTCCCAG ATTCCAGTAA 3180CTCCCAATCT TCTCTCTGCA GAGCCCAAAT CTTGTGACAA AACTCACACA TGCCCACCGT 3240GCCCAGGTAA GCCAGCCCAG GCCTCGCCCT CCAGCTCAAG GCGGGACAGG TGCCCTAGAG 3300TAGCCTGCAT CCAGGGACAG GCCCCAGCCG GGTGCTGACA CGTCCACCTC CATCTCTTCC 3360TCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGAC 3420ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAA 3480GACCCTGAGG TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACA 3540AAGCCGCGGG AGGAGCAGTA CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG 3600CACCAGGACT GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA 3660GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGTGGGA CCCGTGGGGT GCGAGGGCCA 3720CATGGACAGA GGCCGGCTCG GCCCACCCTC TGCCCTGAGA GTGACCGCTG TACCAACCTC 3780TGTCCCTACA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA 3840GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT 3900CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT 3960GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG 4020GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC 4080GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA ATGAGTGCGA CGGCCGCGAC TCTAGAGGAT 4140CTTTGTGAAG GAACCTTACT TCTGTGGTGT GACATAATTG GACAAACTAC CTACAGAGAT 4200TTAAAGCTCT AAGGTAAATA TAAAATTTTT AAGTGTATAA TGTGTTAAAC TACTGATTCT 4260AATTGTTTGT GTATTTTAGA TTCCAACCTA TGGAACTGAT GAATGGGAGC AGTGGTGGAA 4320TGCCTTTAAT GAGGAAAACC TGTTTTGCTC AGAAGAAATG CCATCTAGTG ATGATGAGGC 4380TACTGCTGAC TCTCAACATT CTACTCCTCC AAAAAAGAAG AGAAAGGTAG AAGACCCCAA 4440GGACTTTCCT TCAGAATTGC TAAGTTTTTT GAGTCATGCT GTGTTTAGTA ATAGAACTCT 4500TGCTTGCTTT GCTATTTACA CCACAAAGGA AAAAGCTGCA CTGCTATACA AGAAAATTAT 4560GGAAAAATAT TCTGTAACCT TTATAAGTAG GCATAACAGT TATAATCATA ACATACTGTT 4620TTTTCTTACT CCACACAGGC ATAGAGTGTC TGCTATTAAT AACTATGCTC AAAAATTGTG 4680TACCTTTAGC TTTTTAATTT GTAAAGGGGT TAATAAGGAA TATTTGATGT ATAGTGCCTT 4740GACTAGAGAT CATAATCAGC CATACCACAT TTGTAGAGGT TTTACTTGCT TTAAAAAACC 4800TCCCACACCT CCCCCTGAAC CTGAAACATA AAATGAATGC AATTGTTGTT GTTAACTTGT 4860TTATTGCAGC TTATAATGGT TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG 4920CATTTTTTTC ACTGCATTCT AGTTGTGGTT TGTCCAAACT CATCAATGTA TCTTATCATG 4980TCTGGATCCT GTGGAATGTG TGTCAGTTAG GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG 5040GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG 5100GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCATAGTCC 5160CGCCCCTAAC TCCGCCCATC CCGCCCCTAA CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC 5220ATGGCTGACT AATTTTTTTT ATTTATGCAG AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT 5280TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG GCCTAGGCTT TTGCAAAAAG CTAATTC 5337

We claim:
 1. An isolated DNA sequence encoding a substrate foraggrecanase, wherein said substrate contains, as the only amino acidsequence of aggrecan, the interglobular domain of human aggrecan.
 2. ADNA sequence encoding a substrate according to claim 1, wherein said DNAsequence comprises the nucleotide sequence of nucleotides 2350 to 4114of FIG. 7 (SEQ. ID NO. 4).
 3. A DNA sequence encoding a substrateaccording to claim 1, wherein said DNA sequence comprises a portion ofthe nucleotide sequence of nucleotides 2350 to 4114 of FIG. 7 (SEQ. IDNO. 4).
 4. A DNA sequence encoding a substrate according to claim 1,wherein said DNA sequence hybridizes under stringent conditions with thenucleotide sequence of nucleotides 2350 to 4114 of FIG. 7 (SEQ. ID NO.4).
 5. A vector comprising a DNA sequence as claimed in claim
 1. 6. Ahost cell comprising a vector as claimed in claim
 5. 7. A vectorcomprising the nucleotide sequence set forth in FIG. 7 (SEQ ID NO:4). 8.An isolated DNA sequence encoding a polypeptide substrate foraggrecanase as claimed in claim 1, wherein said substrate comprises aportion of the sequence as set forth in FIG. 6 (SEQ. ID NO. 3).
 9. Anisolated DNA sequence encoding a substrate for aggrecanase, wherein saidsubstrate comprises the following components, beginning with theN-terminus and ending with the C-terminus: a) the signal sequence ofCD5; b) the FLAG-epitope; c) the interglobular domain of human aggrecan;d) the hinge region of human IgG1; e) the CH2 region of human IgG1; andf) the CH3 region of human IgG1.
 10. An isolated DNA sequence encoding apolypeptide substrate for aggrecanase, wherein said substrate comprisesthe amino acid sequence as set forth in FIG. 6 (SEQ. ID NO. 3).
 11. Anisolated DNA sequence encoding a polypeptide substrate for aggrecanase,wherein said substrate comprises the amino acid sequence as set forth inFIG. 6 (SEQ. ID NO. 3) and wherein amino acid 34 is mutated to Ala. 12.An isolated DNA sequence encoding a substrate as claimed in claim 11,wherein said substrate comprises a portion of the amino acid sequence asset forth in FIG. 6 (SEQ. ID NO. 3) and wherein amino acid 34 is mutatedto Ala.
 13. A cell culture system for monitoring aggrecanase activity ina sample comprising: (a) mixing freshly isolated chondrocyte cells and asubstrate for aggrecanase encoded by the DNA of claim 1; (b) incubatingthe reaction mixture of step (a); and (c) detecting the presence orabsence of aggrecanase activity in the reaction mixture, whereinaggrecanase activity is determined by the presence of aggrecanasepeptide cleavage products.
 14. A system as claimed in claim 13, whereinsaid system is free of endogenous proteoglycans or other extracellularcomponents.
 15. A system as claimed in claim 13, wherein the presence orabsence of cleavage products is measured by determining the presence ofpeptide cleavage products that react with monoclonal antibodies specificfor aggrecanase cleavage products.
 16. A system as claimed in claim 15,wherein said monoclonal antibody detects a peptide having an amino acidsequence ARGSV.
 17. A system as claimed in claim 13, wherein aggrecanaseactivity of said chondrocytes is stimulated by adding retinoic acid tothe reaction mixture of step (a).
 18. A method for cloning cDNA encodingaggrecanase, comprising: (a) preparing a cDNA expression library fromcells expressing aggrecanase; (b) transfecting suitable cells with thelibrary of step (a), wherein said cells express said cDNA in saidexpression library; (c) incubating the cells of step (b) with asubstrate for aggrecanase encoded by the DNA of claim 1; and (d)detecting the presence of an aggrecanase cleavage product produced by acell of step (c).
 19. A method for screening for an aggrecanaseinhibitor, comprising: (a) mixing freshly isolated chondrocyte cells anda substrate for aggrecanase encoded by the DNA of claim 1; (b)incubating the reaction mixture of step (a) in the presence or absenceof a putative aggrecanase inhibitor; and (c) detecting the presence orabsence of aggrecanase activity in the reaction mixture whereinaggrecanase activity is determined by the presence of aggrecanasepeptide cleavage products.
 20. A method for monitoring the onset ofosteoarthritis, said method comprising assaying a sample of biologicalfluid from a patient for the presence of aggrecanase, wherein saidaggrecanase activity is measured using a system as claimed in claim 12.21. A method for monitoring the progression of osteoarthritis, saidmethod comprising assaying a sample of biological fluid from a patientsuffering from osteoarthritis for the presence of aggrecanase, whereinsaid aggrecanase activity is measured using a system as claimed in claim13.
 22. A method as claimed in claim 20, wherein said biological fluidis selected from the group consisting of synovial fluid, urine, serum,and lymph fluid.
 23. A method as claimed in claim 21, wherein saidbiological fluid is selected from the group consisting of synovialfluid, urine, serum, and lymph fluid.
 24. A method as claimed in claim21, wherein said method is used to follow disease progression todetermine the effectiveness of therapeutic treatment.